Multiple modes of association in bovine prothrombin and its proteolysis products.

Bovine prothrombin has been demonstrated to selfassociate both in the absence and presence of Ca2+ ions. Prothrombin Fragment 1 (residues 1 to 156 of prothrombin) self-associates in the presence of Ca2+ and other divalent cations, but not in their absence. Prethrombin 1 (residues 157 to 582 of prothrombin) selfassociates indistinguishably both in the absence and presence of Ca2+ ions. Self-association of Prothrombin Fragment 1 in the presence of Mn2+ ions has been shown to be dimerization both by sedimentation equilibrium and sedimentation velocity measurements. Association constants for dimerization induced by M&+, Ca”, and Sr2+ ions have been calculated from sedimentation velocity data. The ability of the divalent cations to induce self-association is in the order Mn” >> Ca” 2 Sr2+ > M&+. A hybrid “dime? between prothrombin and Prothrombin Fragment 1 has been demonstrated to form in the presence of Ca2’ ions. Fluorescence polarization studies have demonstrated that the rotational relaxation time for Prothrombin Fragment 1 in a viscous solution is increased approximately a-fold in the presence of Ca’+, implying that self-association is reflected in the spectrally monitored hydrodynamic properties, as well as sedimentation properties of the protein molecule. The tryptic peptide which contains 8 of the 10 y-carboxyglutamic acid residues of prothrombin (residues 12 to 44) binds divalent cations and precipitates, rather than undergoing the limited self-association which occurs with Prothrombin Fragment 1. On the basis of these observations, the self-association behavior of prothrombin must be concluded to occur via at least two different mechanisms, one dependent on the presence of Ca2’ ions and one independent of Ca2+ ions. The following observations suggest that specific, cooperative Ca2+ binding and Ca”-induced selfassociation require particular structural constraints in the protein molecule. 1) Divalent cations induce precipitation of tryptic peptides from prothrombin. 2) Previous results demonstrated that when the disulfide bridges of Prothrombin Fragment 1 were reduced and